1. Field of the Invention
This invention relates to novel disopyramide derivatives pertaining to immunoassays for determining the drug in liquid media such as biological fluids. Such derivatives include immunogens used to stimulate production of antibodies to disopyramide in host animals by conventional techniques. Also provided are labeled disopyramide conjugates used as reagents, along with the antibodies, in particularly preferred immunoassays. Intermediates in the synthesis of the aforementioned immunogens and labeled conjugates are also provided.
Disopyramide [Merck Index, 9th ed., pp. 449 & 450 (1976)] is a cardiovascular drug of the formula: ##STR1## used to treat advanced coronary artery disease and to prevent specific ventricular rhythm disturbances including those following myocardial infarction. [J. Koch-Weser, New Engl. J. Med. 300: 957 (1979); G. Jennings, et al, Am. J. Cardiol. 46: 469 (1980)]. It is sold in the U.S. as the mono-phosphate salt under the tradename NORPACE.RTM. ("Physicians' Desk Reference", 36th ed., Medical Economics Co., Oradell, NJ, 1982, p. 1784).
Disopyramide also posseses anti-cholinergic properties, and the most common side effects of the drug are due to this aspect of its pharmacological activity. Patients with glaucoma or benign prostatis hypertrophy are at particular risk. Disopyramide can also induce many types of cardiac dysrhythmia, as well as depress myocardial contractility, and cardiac and arterial pressure. Such toxic effects are usually dose-related and are due to excessive concentrations of the drug in the plasma or myocardium [J. Koch-Weser, supra; E. G. Manolas, et al, Med. J. Aust. 20 (1979)].
Therapeutic plasma levels occur in a narrow concentration range of 2-4 micrograms/mL [P. Danilo and M. R. Rosen, Am. Heart J. 92: 532 (1976)]. The half-life of disopyramide in normal volunteers ranges from 3.4 to 10.8 hours. Such variations in half-life make measurement of plasma levels essential to ensure that the dosage is appropriate to maintain plasma levels within the therapeutic range, and to decrease the side effects associated with higher levels of the drug [K. Ilett, et al, J. Chromat. 154: 325 (1978)].
2. Description of the Prior Art
Several analytical methods have been developed for measuring blood levels of disopyramide and its principal metabolite. They include gas-liquid chromatography [A. Johnston and D. McHaffie, J. Chromat. 152: 501 (1978)], high performance liquid chromatography [P. -O. Lagerstrom and B. -A. Persson, J. Chromat. 149: 331 (1978)], thin layer chromatography [R. N. Gupta, et al, Anal. Chem. 51: 445 (1979)], enzyme-mediated immunoassay [A. Johnston and J. Hamer, Clin. Chem. 27: 353 (1981)], and differential-pulse polarography [J. F. Burmicz, et al, Analyst (London) 106: 802 (1981)].
The preparation of antibodies to disopyramide for use in immunoassays to determine the drug has been accomplished in the prior art by essentially two different approaches. One approach has been to couple the drug through its amide group to an appropriate carrier material [U.S. Pat. No. 4,288,553]. The second approach involves random nitration of the phenyl ring followed by conversion of the nitro groups to amino groups and conventional peptide condensation to the carrier molecule [Japanese Provisional Spec. No. 56-046,820 (Derwent No. 44742D/25)]. The immunogens resulting from the second approach are a mixture of ortho-, meta-, and para-coupled drug derivatives producing a particularly heterogeneous antibody population.
The state-of-the-art of preparing antibodies to haptens such as drugs is represented by Weinryb et al, Drug Metabolism Reviews 10: 271 (1979); Playfair et al, Br. Med. Bull. 30: 24 (1974); Broughton et al, Clin. Chem. 22: 726 (1976); and Butler, J. Immunol. Meth. 7: 1 (1976) and Parmacol. Rev. 29(2): 103 (1978). Labeled conjugates, comprising the analyte or a derivative or other analog thereof, coupled to a labeling substance are variously described in the literature, e.g., U.S. Pat. Nos. 4,279,992; 4,182,856; 4,259,233; and 4,292,425 wherein the label is the fluorogenic enzyme substrate .beta.-galactosyl-umbelliferone.